Method of propagating yeasts



Patented Sept. 13, 1927.

UNITED STATES ARNOLD K. BALLS, OF PHILADELPHIA, PENNSYLVANIA.

METHOD PBOPAGATING YEASTS.

No Drawing.

This invention relates to the manufacture of yeast for baking and otherpurposes by the use of a particular race of yeast, and more particularlyto the production of yeast in very large yields, of good color, keep ngqualities, and baking strength, by us ng chiefly as raw material crudeWest Indian molasses which has never before been suc cessfully used inyeast manufacture (except for very low' yields) and also by using a newrace of yeast whereby these novel results are obtained.

This organism is apparently a new species of Saccharomyces and wasisolated by well known bacteriological means from a fermenting molassessolution. It is reasonable to suppose that the yeast in question has hadan environment of cane molasses for many generations in the past, andhas thereby acac quired certain tolerances for constituents of crudemolasses and waste syrups, which are not possessed by Whether thesedifferences are sufiicient from a purely botanical standpoint toclassify this yeast as a new species or not, they are ample todifferentiate it markedly from any other known race of yeast in regardto rate of growth and in particular, its behaviour in cane molasses.

It has been shown, princi ally by Slator- Biochem. Jour. vii (1913), tat in a definite medium, a yeast grows accordin to a definite law, theso-called logarithmic law, represented by EulerChemie der Hefe, p. 253,in Briggs logarithms by the expression in which K is a constant, soderived as to be a positive number, a is the amount of 4 yeast at thebeginning, and a+w the amount of yeast at the end of growth.The'signifi-. cance of K is given also by Euler-ibid, p. 255as followsthe growth constant K=0.4343K=% (log (a-l-x) log (a)) is then an exactmeasure of the speed of growth. Growth finally becomes impeded due tolack of food, the toxic eflect of the accumulation of various metabolicproducts, etc. The constant, however, when determined on a species ofyeast during that period of the g owth cycle when growth is unimpededshows the speed at which the yeast is capable u of growing in aparticular medium.

other Saccharomyces.

Application filed July 18,1888. Serial No. 852,455.

Different yeasts are capable of growing in a medium such as molasses forinstance, at different rates of speed, which, since the medium is thesame, depend solely upon the reproductive ability of the yeast, that isto say, upon a quality inherent in the yeast cell itself. The possessionof a low, or of a high, reproductive ability under the circumstances, isas much a definite characteristic of the yeast in question as the shapeof the cells, and more definite than the ability to ferment mannose orlactose. The organism which I have isolated, and

'which is an integral part of this invention,

because without its use the same strikin results cannot be obtained, ischaracterize by having a higher growth constant in molasses media, andparticularly in cane molasses media, than any of the other organismstested by me, which includes all the ordinarily used species of alcoholforming yeasts, whose growths can be successfully removed from theliquid by filtration, centrifugation, and the like known processes.There are yeasts whose growths are so slimy that it is not practical toremove them from solutions. Such yeasts have no practical value at thistime and are not included in the scope of this work.

This yeast forms no spores, no scum, no marked sedimentation, and itscytological characteristics are those of an ovoid yeast of theSaccharomysec genus reproducing by budding. The mor hologicalcharacteristics are not specific or 'stinguishing except the ablillityof the yeast to grow as .separate ce s.

An experiment, for example, comparing this new yeast with one of thebest growing known races in use S. eerecisz'w, under exactly similarconditions in a medium of diluted molasses, ammonium hosphate andammonium sulfate, showed va ues of Kthe growth constant of Euler-asfollows:

In beet molasses. In cane molasses.

Commonly yeast.

New

Commonly W yeast.

used

Hours 0! growth.

i yeast.

New yeast.

These fi es show not only the greaterrapidity oi g'rowt-h of the newyeast, but

also th'e fact thatit grows as well on cane molasses without anclarificationor special separate instead of tion of growt Since thesynthesis of protein requires n1trotreatment as the ot er yeast grows onbeet molasses.

In addition to the above methods of dif ferentiating this yeast from thevarieties used heretofore, it may also" be distinguished bythe fact thatduring itsentire period of growth, it is always very" finely divided andshows very little. tendency to settle or sediment. Under-the microscope,the cells are seen to be all separate except where an undevelopeddaughter cell has not dividedofi from the parent. Even in unstirredsolutions and on solid media, this tendency to owing in clumps "andchains is marked an .enables the east to'be easily distinguished aftera; litt e practice Y from the various races 6f 8; cereoisc'w which atthe outset of any stage of multiplication,-

grow in chains or m clum s.

Because of the peculiarity of this characteristic and its' robablesignificance on the ,owth rate, it as been proposed to name $5 yeast15'. disjwnoms-sp. nov. The fact that the cells are so separated allowsthe entire membrane of each cell to be completely surrounded by a layerof the nutrient solution, which accounts no, doubt in some degree, forthe greater metabolic activity of the species.

This increased metabolic activit is evidenced on the art of theyeast int e direcrather than fermentation.

gen, one might expect the nitrogen consumption of this yeast to begreater than that of S. cerem'siaa. My experlments have shown that theyeast can withdraw ammonium nitrogen from the medium in about twice theamount used by S. cere'vz'sz'w under the same conditions. The largequantities of nitrogen if added in the form of highly ionized ammoniumhydroxid and ammonium salts at the start of a fermentation would produceundesirable osmotic effects, consequently, it is recommended to addthese, as is often done, periodically during the life of the mash,either mixing in with freshly added molasses, or separately. The precisetimes and quantities always must be left to the discretion of theoperator and it is impossiblev that other processes do; that 1s, ayeastoi.

superior multiplication ability prepared by a special and expensive mashusually from grain. In the process of this mventlon, there is no stockmash, the seed for the fermenter being grown in steps from the originalculture in quantities adapted to ro, production of not less than thaneight times the seed used inthis step. Thesesteps, therefore, are allalike, except in regard to the quantities of the materials taken I havefound-that this race of yeast possesses the peculiar propert ofpreventing uce a rethe growth of organisms o the mycederma yeast typeand ver often kills them off. The yeast 8. tested, in comparison with.other yeasts to determine its fermenting characteristics on variousslugars, and has been found to differ from a ing properties. The otheryeasts employed in this test were 8. cereois'iw Frohberg type,Hansen,Saaz ty e,'Ru perts yeast, anEnglish ale yeast, ace II, S. logos,S. pastorianus apwwlams' S. 'elhlpsoz'deus II and S. I aZ' us. "All ofthe yeasts em:

ployed. were cultivated for a period of nine months in saccharose, and 1all the specimens ferment saccharose and dextrose.

The most characteristic difierence between the present yeast and otherknown yeasts, from a fermenting standpoint, is its ability to fermentinosite (a cyclo-hexane derivative. Inosite is commonly used in the our,or more as'junctus has also been. i

l of these other yeasts in its fermentsame manner as true sugars indetermining the fermenting characteristics of microorganisms. This, asfar as I know, is unique among such organisms. None of the other knownyeasts emplo ed in the experiments has the property of ermentinginosite. All of the yeasts employed in the experiments will fermentfructose and mannose, and most of them will ferment maltose andraifinose. 1S. disjumctus has the roperty of fermenting all of thesesugars and has the further property of fermenting galactose, which ispossessed by only four other yeasts, namely,

Frohberg type, Race XII, logos, and pasto'r'z'anus. None of the yeastsemployed, in-

cludingS. disjqmotus, would ferment a solution of penta acetyl glucose,and logos and (Zisjumom were the only yeasts capable of fermenting asolution of alpha methyl glucoside.

I have also conducted experiments to identify the yeast 8. disjunorus byprecipitin tests, which is simply an a plication of the well knownmethodsof di erentiating bacteria, to yeast. 4 i i 1 The serum isprepared by injecting a suspension of yeast cells into a rabbit atvarious intervals, then bleedin the 'rabbit,,and

rom the clotted.

removing the clear serum blood. v

In the following tables are shown the results obtained from thetreatment of known yeasts with a serum of S. disjmotm yeast and also theuse of sera from the known yeasts and the culture of S. disjunctus. Themade. In the table, a single cross represents tests are specific inshowin that S. dz'sa slight reaction,'two or three crosses heavierjunctus reacts strongly with its own serum reactions, and four crosses'avery heavy reonly and that other sera do not react with action. Thefirst table shows the results 10 i the culture ofS. disjunctus, althoughreactobtained from tests conducted on well known ing with the yeast fromwhich they were commercial yeasts:

O O O O C o o O O O E t2 0 o c c o o o o i f i o o o c o Q O O Q Q 0 O aO Q a g E 1 o o c o c c i Q g 1 c o o o o i e V c o o c o o c o i E a a,2 E i o c i c i i g i i i i g l ,g c

a, E o o c c: c o c g Q 0 Q O c N g a i i i i i i i v5 i E E i i i 5 a 5a a z a a g; a a E 1 g "g 1. a 5 s a a a E a 2 S a as s No: 3 o i g 5 EH3 3 o E El 33 o S m 1 H m mixed with it, Callahan is the same and so on.It is to benoted none of them correspond to S. dz'sy'umtus. s

The following table shows the results obtained when a serum of disjmwtueis added to a solution of other yeast and also the results obtained whensera of other yeasts and disjunctus yeast are added to the other yeast.

Serum diajunctus with other yeast- Other sera with disj'unctut yeast.

Dilution. a; tie 1in a. 11kt) 0 a rt-c a a n 0 Validua .2 0 0 mar 0 bApiculatua 0 0 0 0 Paatorlanus 0 0 0 0 Saaz 0 0 o Frohberg o 0 Hansen oo o o o "PP"'S -l-+ 0 o o I A1eyeaa-.-. I o o 0 Race x11 o The othersera referred to in the heading in the second column in the above tableare prepared-from the yeast-s specified in horizontal rows to the left.

In growing yeast according to the modern rocesses, aeration is employedand this s requently accompanied by the growth of other organisms, suchas wild yeast and bacteria, many of which are very detrimental to theculture yeast itself. Various methods of lessening this dan er areknown, such as cleanin partial sterilization by heating, or by additionof antiseptics and the like (see the patent to Hayduck, No. 1,449,112).However, in spite of these precautions, the danger still remains, and asair itself quite often contains members of the so-called mycodermayc'astgroup which are among the commonest as well as the most dangerous infections in heavily aerated mashes where the acidity is low, the generaltendency of the more modern practices is to lead to the formation andgrowth of mycoderma yeast.

The saving in labor andattention to the mash and the added certainty ofobtaining good yeast are obvious advantages. This is especially true ofyeast to be used in repropagation, that is, stock yeast, because aninfection of the stock is usually multiplied many times in thesucceeding mash.

To prevent growth of organisms of the 4 mycoderma yeast type, smallproportions of this race of yeast may be mixed with stock yeast to beused in any process of yeast propagation and upon growing the mixedculture, it will be found that mycoderma 4 yeast present do not multiplybut on the other hand, generally decrease in amount.

This yeast may be employed in a mixture with yeasts now generally emploed for baking purposes without deleterious y eflect- 5 ing the color,yield, bakin strength or keeping quality of the yeast. lhe amount ofadmixture of the new yeast to be used ma vary within wide limits, aslittle as one-fi teenth of the total stock being found suifiq cient toprevent the growth of mycoderma yeast and as much as one-half may be.used without injurious action on the product.

As an example, the following experiment was performedf 'Four aeratingjars containing the same amounts of a molasses medium were heavily in-.fected with a suspension of mycoderma yeast, and to each was added asstock, different proportionsof a well known baking yeast and 'nComposition of the stock yeast.

Co. of a suspension in water. Jar A. Jar B. Jar C. Jar D.

8.c1n.3g.per50cc 115 125 12.5 12.5 New race 3 g. per 50 cc 12. 6. 25 12.5 Mycodcrma yeast 10 .10

Cell counts during the experiment:

[Average count-cells per 1-4000 cu. mm. liquid] Hour of experiment. A.B. C. D.

Of the pairs of figures, the firstfigure is the number of mycodermacells, the second the number of baking yeast cells.

This new yeast diflers from known yeasts not only in growing better inmolasses, but

by growing with the production of. a white east of ood baking qualitiesin the most ighly co ored and impure varieties of molasses, particularlythat grade known as crude Cuban cane, which in the trade usuallydesignates any low grade West Indian molasses, or sugar house waste.Such Cuban cane molasses is black in color and contains many substancesof phenolic structure inhibitory and even toxic to other yeasts. It ismuch darker and more impure than the socalledrefiners molasses on whichthe com-' mercial rowth of yeast has been claimed. It is so iflerentfrom the molasses of the prior'art as to be considered an entirelydifferent substance.

From Cuban cane molasses, a yieldvarying from fifty to one hundred percent of the weight of the molasses can be obtained of good bakingyeast/having excellent color and eeping qualities, without anypreliminary purification or clarification of the molasses, using thisnew culture and process.

Due to the inhibiting act-ion on most bacteria and moulds of some of thecane molasses constituents remaining in the ressed yeast, this yeastkeeps very well. It IS notale, for exceptional viability on drying forthe same reasons.

In practicing the process, the yeast may be (propagated by any of theusual methods an I prefer to employ crude West Indian molasses wheneverpossible on account of its 6 relative cheapness. The provision of largerquantities of nitrogen than is usually necessary in the propagation ofyeast may be obtained by the use of ammonia, ammonium salts or otheramino 'or amido compounds. When good bakin qualities are desired, the

propagation should be controlled by pre-' venting multiplicationexceeding eight or ten times the stock added. Phosphoric acid in somesimple form, such as phosphate, ma

be added in an amount commensurate Wit the yield expected and the commonpractice of fermenting in a weak acid solution should be adhered to. Theacidity of the solution may be determined in the usual manner by the useof litmus paper or hydrogen ion measurements and ammonia or other alkaliadded .to the solution at intervals in an amount sufiicient to reduceits acidity without rendering the fermenting solution alkaline. Ifalkalinity appears impending in the mash, a suflicientamountof sulfuricacid or other acid may be added to render it suificiently acid. Theusual separating, pressing, and subsequent handling steps now employedin common practice may be resorted to in separating the yeast from thesolution and placing it in proper condition for marketing or otherhandl1ng.-

The baking quality of this yeast may be further improved if a bakingyeast is desired rather than a yeast for fertilizer, stock food and thelike, to which the process is equally applicable. This im rovement isobtained by restrictin the ma tiplication of the yeast and not allowingan increase of more than eight or ten times the original stock in asingle fermentation. If the multiplicationpf the yeast is notrestricted, its

emphatic 'tendency to reproduce at all hazards causes the cells tobecome weakened. As an example of the most approved form of the process,the following is given:

One hundred pounds of crude West Indian cane molasses is diluted toabout four times its bulk with water, heated to approximately 70 C., andfiltered if any solid dbris must be removed. About'ten per cent of thefiltrate is placed in the fermenter and diluted to about one hundredgallons with water. The yeast is added to the fermenter in amount fromfive to twenty per cent of the total molasses, but for good bakingyeast, about fifteen per cent is best. From one to two pounds bfammonium phosphate and about half thatquantity of ammonium sulfate isalso added to the fermeutcr, which is maintained at as near 28 C. aspossible. A heavy current of air isset up and aqua ammonia, preferablydiluted, al lowed to run in slowly at such a rate that the acidity doesnot drop materially below that equivalent to.0.05 cc. of normal solutionper 10 cc. mash. The rate should be varie by the operator during theprocess. The ammonia is added continuously until between five and sevenpounds of strong aqua ammonia are added, depending upon the amount ofair used. This should take eight to twelve hours. The remaining molassesis diluted to one hundredgallons with water and run in a constant streaminto the fermcnter, at such a rate that its addition will occupy allbuttwo hours of the time desired to run the mash. The total mash time isusually about fifteen hours, but may be as long as twenty hours. Withthese quantities, no attention to the gravity is needed as this makesvery little difference. The customary practice in raising the acidity atthe end of the fermentatiomand in regard to separating, cooling, etc.,is followed. From such a mash, a total yield of greater than the weightof the molasses taken should be easily obtained, using the organismdescribed. The baking strength, color, and sugar-splitting activity willbe good.

I do not wish to restrict the process to the use of crude cane molassestothe exclusion of refiners molasses or beet molasses, or any othersubstances known to favor yeast growth, such as, for instance, grains,malts,

the steep water of corn, as disclosed in the patent to Wagner, No.1,434,462, and the -various nitrogenous wastes of slaughter houses and ae, the aci extract of bones and the like, a of which furnish growth ofyeast.

While the newly isolated yeast has various advantages, its ability togrow in cane molasses and especially the crude Cuban cane molasses isperhaps its most marked characteristic and the most importantcharacteristic from a practical standpoint as it permits the use of thisrelatively cheap raw material in the manufacture of yeast. on a scalethat compares favorably with the growth of other yeasts in beetmolasses.

While I have described in detail the preferred practice of my process,it is to be understood that the details of procedure may be widelyvaried without departing from the spirit of the invention or the sco eof the subjoined claims.

aving thus described my invention, I

claim: g a

1. The method of growing yeast which 60 comprises adding a yeast stockof Sascharomycea dz'jwnctus to a mash of crude West Indian cane molassesand allowing theyeast to propagate therein.

2. A rocess of propagating Sacckaro- Q6 myces dzsy'wrwtus whlchcomprises inoculating an aqueous solution of crude West Indian molassescontaining a compound containing the phosphate radical soluble in thesame and a water soluble salt containing the good material for thepacking houses, including tankammonium radical with Saccharomycesdiszmctus and propagating the Sacoharomyces disjwnctus in such solution.

3. A process of propagating Sacckaromyces dzsjunctus which comprisesinoculating a yeast propagating aqueous solutior favorable to thedevelopment of such yeast with Sacclzaromg ces disjunctua, said solutioncontainin a compound containing the phosphate radical soluble in thesame and water soluble salt containing theammoniurr radical andcarbamid, and propagating the Saccharomyces disjunctus in such solution.

4. A method of retarding growth of mycoderma yeast during propagation ofa yeast of the Saccharomyces family which comprises inoculating a yeastpropagating solution with Sacckaromg ces disjumim and a yeast of theSaccharomyces family other than Saccluu'omyces disjunctus and allowingthe last two mentioned added yeasts to propagate therein.

5. A method of retarding growth of mycoderma yeast during propagation ofa yeast of the Saccharomyces family which comprises inoculating a yeastpropagating solution-with a yeast of the Saccharomyces family other thanSaccblaromyces disjunctus containing from 10 to er cent of a culture ofSascharomyces zsjwrwtusand allowingthe inoculating yeasts to propagatetherein.

6. A method of retarding growthof myvcoderma yeast during propagation ofa yeast of the Saccharomyces family which comprises inoculating a yeastpropagating solution of crude West Indian molasses with Saccharomycesdz'ay'mwtus and a yeast of the Saccharomyces family other thanSaccharmn/yces disjumctw: and allowing the last two mentioned addedyeasts to propagate thgreip. f p S M rocess 0 r0 ting acc r0- m/yces d'jamctwa which bb n iprises inoculating a yeast propagating aqueoussolution of crude West Indian molasses containing a water solublecompound containing the phosphate radical and a .Water soluble saltcontaining the ammonium radical with Saccharom/yces dz'sy'emctus,propagating the Sacckarom/yces disj'zmctus m such solution, and addingthereto during the propagation additional amounts of crude West Indianmolasses, the water soluble com ound containin the phosphate radical anthe water solub le salt containing the ammonium radical.

In testimony whereof, I aflix my signature.

ARNOLD -K. BALLS.

